The antigen-specific cytotoxicity mediated by cytolytic T lymphocytes (CTL) is an important part of immune responses. It is the ultimate goal of our studies to reveal the cellular and molecular requirements for CTL effector functions and differentiation. Our studies of the regulation of T lymphocyte functions by protein phosphorylation are divided into experiments designed i) to test the ectophosphorylation model and ii) to continue the exploration of the role of intracellular protein kinase A (PKA) and PP2a protein phosphatase in CTL generation and functions. A) Ectophosphorylation Model of T Cell Development and Effector Functions. According to this model the phosphorylation of extracellular domains of cell surface proteins affects cognate lymphocyte cell-cell interactions. It is assumed that the ligand specificity of cell-surface receptors (cell adhesion proteins, recognition molecules [e.g. TCR on T cells, sIg on B cells]) could be regulated through phosphorylation of their extracellular domains in a manner now accepted as a mechanism for the regulation of enzyme-substrate interactions. Use of a panel of monoclonal antibodies and a gamma-32P-ATP ectophosphorylation assay allowed us to demonstrate the ectophosphorylation of several important surface antigens including TCR, T200, and HSA. We demonstrated the location of the phosphorylated site in the TCR ectodomain using transfectants with GPI-linked T-cell receptor. These data point to the need to evaluate the ecto-phosphorylation event as potentially regulating TCR affinity for antigen. Work is in progress to determine the exact location of the ecto-phosphorylation site. We have shown that ectoprotein phosphatase activity could be explained by release of the intracellular PP2a enzyme. Experimental tools (cDNA constructs, antisense mRNA oligos) for suspected ecto-enzymes including casein II protein kinase are being developed to directly investigate the role of these proteins in T cell functions. B) Studies of Intracellular Protein Kinase A and PP2a Protein Phosphatase. Transgenic mice with thymus-specific expression of PKA inhibiting the RIalpham subunit have been developed for use in studies of the role of PKA in T cell differentiation. Studies of the PP2a phosphatase led to the demonstration of PP2a association with the plasma membrane in complex with a small subset of high-affinity Con A-binding proteins.